的蛋白质 - 蛋白质相互作用抑制剂的发展，通过替换：应用程序的设计和开发非ATP竞争性CDK抑制剂
Summary
We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.
Abstract
REPLACE是发展到更有效地针对蛋白质 – 蛋白质相互作用（质子泵抑制剂）一个独特的策略。其目的是通过对抑制剂的识别这样的结合位点并提供改进的方法代表了大多数潜在的药物靶点，扩大可用药物靶点的空间。本文的主要目的是提供替换策略，涉及的计算和合成化学方法的使用和应用的方法概述。取而代之的是通过其应用到非ATP竞争性细胞周期蛋白依赖性激酶（CDK）抑制剂作为抗肿瘤治疗学的发展例举。激酶经常放松管制在癌症，因此被认为是药物开发的重要目标。在S期CDK2 /细胞周期蛋白A的抑制已报道通过E2F1途径促进癌细胞中p53的独立的方式选择性凋亡。靶向蛋白质 – 蛋白质相互作用的细胞周期蛋白结合蛋白摹槽（CBG）是一种方法，这将使细胞周期在转录激酶特异性抑制。该CBG是由CDK底物和肿瘤抑制蛋白的共识序列中，识别被称为细胞周期蛋白结合基序（CBM）。煤层气先前已优化，从P21WAF（HAKRRIF），然后八肽进一步截短为五肽保持足够的活性（RRLIF）。在一般不细胞渗透性肽，是代谢不稳定，因此，策略REPLACE（通过计算富集代用偏配体替代品）已被以产生更多的类药性的抑制剂的应用。策略开头片段结扎抑制肽（翻转），其选择性地抑制细胞周期CDK /细胞周期蛋白复合物的设计。通过迭代地更换HAKRRLIF / RRLIF的残基片段像小分子（封端基团），从N末端（NCAPS）开始生成翻转，随后更换上C末端。这些化合物开始的非ATP竞争性CDK抑制剂作为抗肿瘤疗法的产生点。
Introduction
在这篇文章中，应用REPLACE（更换使用计算富集部分配体的替代品）策略，以蛋白质-蛋白质相互作用的肽酶抑制剂转换成多种药物相关的分子为例进行说明1-3。虽然生产者价格指数代表了丰富，但开发不足潜在的药物靶点的来源，现有方法在很大程度上不足以让这些普及。当前策略包括基于片段设计4，高通量筛选5和6提供了进步装订肽，但是这些在许多情况下是无效的。其结果是，更多的进展和更有效的方法是必需的。 REPLACE已激酶抑制剂具有改善的类药性的属性和有潜力的进一步发展为抗肿瘤治疗剂的开发已充分验证。这种策略是例示的电池的非ATP酶抑制剂的开发周期CDK的并涉及如下：1）获得关于HAKRRLIF / RRLIF与细胞周期蛋白结合槽相互作用三维结构信息; 2）确定的重要结合决定肽相互作用;含有一种或多种结合决定簇的肽的N-末端的3）截断; 4）计算确定可能的小分子的替代品（局部的配位体的替代品，聚乳酸类）的肽和其中的截断部分保留母体肽的关键相互作用; 5）合成或PLA中的商业采购预测与先前所删除的肽残基所占据的子站点贪婪地结合; 6）的合成通过使用固相合成截短的肽最好PLA中的结扎翻转的; 7）检测结合在体外翻转或功能测定（在CDK /细胞周期蛋白上下文荧光偏振），随后在细胞生存力测定法进一步表征。更换的战略中一个示意图y为如图 1所示。在这篇文章中，REPLACE策略的迭代被讨论，并应用到CDK2 /细胞周期蛋白A进行详细说明。激酶被认为是直接或间接地解除管制在大多数肿瘤中，因此被视为适当的抗癌药物靶 7。 CDK的要求协会细胞周期的完全激活，随后磷酸化参与细胞周期调控8关键蛋白。激酶的两个主要群体是控制细胞周期检查站同型[G1 / S（CDK4 /细胞周期蛋白D，CDK6 /细胞周期蛋白D和CDK4 / cyclin E的），S期（CDK2 /细胞周期蛋白A）和G2 / M（CDK1 /细胞周期蛋白B）]和RNA聚合酶通过磷酸化调节（CDK7 /细胞周期蛋白H，CDK8 /周期蛋白C，CDK9 /周期蛋白T）。当E2F1转录因子形成与DP蛋白质然后与DNA结合，并启动基因转录的复合S期进展中的关键步骤发生。 CDK2 /细胞周期蛋白A需要中和E2F1转录通过磷酸化活性，从而导致释放E2F1-DP的复杂和其随后的退化。 CDK2 /细胞周期蛋白A的抑制被认为是维持E2F1在其DNA绑定状态，导致持续激活。的E2F-1的活性得到的水平将超过诱导的p53依赖的细胞凋亡，因此暗示治疗策略所需的阈值。由于E2F-1的失调p53和pRb的通路，高含量经常发生在肿瘤细胞中和抑制CDK2 /细胞周期蛋白A的应导致选择性凋亡的肿瘤和可以被认为是一个有效的癌症靶7。
临床研究CDK抑制剂靶向高度保守的ATP结合位点，导致跨越大于500蛋白激酶在人激酶组之间的反应性和潜在地引起副作用和毒性9。另一种方法是非ATP竞争抑制通过在CBG靶向性底招聘存在于细胞周期正调控亚基并因此显着，并从遥远的ATP结合位点10,11。所述CBG主要是存在于细胞周期蛋白A，细胞周期蛋白D和cyclin E疏水槽并已被证明为识别在基片和肿瘤抑制发现的共有序列。作为一种分离的肽，细胞周期蛋白结合基序（CBM）的结合到CBG，并已显示出抑制的细胞周期的CDKs激酶活性。煤层气已经被优化到八肽（HAKRRLIF，CDK2 /细胞周期蛋白A的IC 50 0.07±0.02μM，CDK4 /细胞周期蛋白D，IC 50 0.88±0.34μM），进而截断为代表的分子量与毒品之间的良好折衷五肽肖像和效力（RRLIF，CDK2 /细胞周期蛋白A的IC 50 1.01±0.17μM， CDK4 /细胞周期蛋白D，IC 50 25.12±2.97μM）12,13。该CBGs由一个大的初级和二级小疏水口袋它是由一个酸化桥接的C区（包括Glu220，Glu224和Asp283）。 HAKRRLIF的关键结合决定簇包括ALA2与Lys3，精氨酸4和Arg5的二次疏水口袋，离子配对和氢键与酸性区域和高度Leu6和Phe8的互补性与亲脂性主要部位的相互作用。此外，大量的氢键被从肽骨架贡献而Ile7充当间隔物残基允许与主口袋最佳的接触。装订模式和HAKRRLIF与CBG的相互作用如图2。
靶向CBM / CBG蛋白-蛋白相互作用将抑制CDK2 /细胞周期蛋白A，CDK2 /细胞周期蛋白E与CDK4 /细胞周期蛋白D的激酶活性，这一点应引起E2F1癌细胞介导的凋亡，而不影响正常细胞7。虽然煤层气衍生肽是细胞周期CDK的有效抑制剂，这是不可能的，他们将作为药物有用，因为它们的代谢不稳定和普遍缺乏的细胞通透性。为此，我们已经为这些有效的肽酶抑制剂转化为更多的类似药物的化合物的抗肿瘤治疗通过CDK2 /细胞周期蛋白利用放松管制E2F1一个抑制进一步的发展应用替换策略。下面的协议，总结了已更换的周期槽的应用程序已经完成的工作。在第一个实例，类药性封端基团替代HAKRRLIF的N末端四肽进行鉴定。此外改善这些群体进行了调查，一个额外的验证研究更换。从这些研究中有代表性的结果也列。
Protocol
Representative Results
Discussion
Targeting protein-protein interactions (PPI) in drug discovery is highly challenging as these typically involve a large shallow contact interface comprised of numerous and diffuse contacts19. Furthermore, peptidic compounds which inhibit PPI’s that are amenable to drug discovery are problematic due to their higher molecular mass, metabolic instability and poor bioavailability20. Current strategies that have been applied for the development of PPI inhibitors include design of proteomimetics and…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Dr’s. Douglas Pittman and Michael Wyatt for their assistance with cell culture and Dr Wyatt and Ms. Erin Anderson for help in development of the binding assays. We acknowledge Mike Walla and Bill Cotham in the Department of Chemistry and Biochemistry at the University of South Carolina for assistance with Mass Spectrometry, Helga Cohen and Dr. Perry Pellechia for NMR spectrometry. This work was funded by the National Institutes of Health through the research project grant, 5R01CA131368.
Materials
| Computational Chemistry | |||
| Accelyrs Discovery studio 3.0 | |||
| Dell Optiplex Workstations | |||
| Synthetic Organic Chemistry | |||
| Silica gel (GF-254 plates) for TLC, Biotage (Uppsala, Sweden) for flash chromatography, Waters Alliance 2695 HPLC with a 2996 diode-array detector and equipped with a C18 (2) 100 A, 250 x 4.6mm, 5μm column (Phenomenox Luna) for purity determination, 1H NMR and 13C NMR spectra were recorded with a Varian Mercury 300 and 400 Spectrometer, respectively. Mass spectra were measured with a Micromass QTOF (Tandem quadruple-1 time of flight mass spectrometer), electrospray ionization (ESI) and VG 70S (Double-focusing magnetic sector mass spectrometer, EI). | |||
| Flourescence Polarization Assay | |||
| 384 micro well plates, , Micro pipets, | Grenier Bio-one | 110256602 | |
| CDK4D1 and CDK2CA (well purified recombinant human kinase complex) | BPS Bio Sciences | 40094(CDK4/Cyclin D), 41101(CDK2/Cyclin A) | |
| assay buffer (25 nM HEPES pH 7, 10 mM NaCl, 0.01% Nonidet P-40, 1mM dithiothretiol (DTT), | |||
| 25 nM HEPES | CALBIOCHEM | 375368 | |
| NaCl | Fisher | 127838 | |
| Nonidet P-40 | US Biological | N3500 | |
| DTT | Aldrich | ||
| -70c freezer | Revco (Ultima II) | ||
| DTX880 multimode detector fitted with 485 nm/535 nm excitation/emission filters and a dichroic mirror suitable for fluorescein | Beckman Coulter, Brea, CA | ||
| Cell Culture | |||
| 96 well plates, | Fisher | ||
| Frozen stocks of U2OS (osteosarcoma) and DU145 (prostate cancer) cell lines | ATCC | ||
| NU serum, DMEM media, trypsin, PEN/STRIP, MTT reagent, | Fisher, Life technology, Alfa Aesar | ||
| Heamocytometer | VWR | ||
| -70c freezer | Revco (Ultima II) | ||
| Incubator | Thermo electron corporation | ||
| Centrifuge | Eppendorf | 5804 R | |
| Refrigerator 4-8C | Isotemp Fisher | ||
| DTX880 multimode detector fitted with 595nm filter. | Beckman Coulter, Brea, CA | ||
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